The publications and other materials are used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
A great variety of assays based on bioaffinity or enzymatically catalyzed reactions have been developed in order to analyze biologically important compounds from various biological samples (such as serum, blood, plasma, saliva, urine, feces, seminal plasma, sweat, liquor, amniotic fluid, tissue homogenate, ascites, etc.), samples in environmental studies (natural, raw and waste water, soil samples) industrial processes (process solutions, products and side products) and compound libraries (screening libraries which may comprise organic compounds, inorganic compounds, natural products, extracts of biological sources, biological proteins, peptides, or nucleotides, and so on). These association assays rely on specific bioaffinity recognition reactions, in which natural biological binding components form the specific binding assay (using biological binding components such as antibodies, natural hormone binding proteins, lectins, enzymes, receptors, DNA, RNA or peptide nucleic acids (PNA)) or artificially produced binding compounds like genetically or chemically engineered antibodies, molded plastic imprint (molecular imprinting) and so on. Such assays generally rely on a label to quantitate the formed complexes after recognition and binding reactions and suitable separation. To achieve efficient separation of bound reagents from unbound components separations such as precipitation and centrifugation, filtration, affinity collection (to e.g. plastic surfaces of tubes, slides or microbeads), solvent extraction, gel filtration or other chromatographic systems are used. The quantitation of the label in free or bound fraction enables the calculation of the analyte in the sample directly or indirectly, generally through use of a set of standards to which unknown samples are compared.
Dissociation assays are assays where, for example specific enzymes catalyze a biological reaction such as hydrolyzing a substrate, transferring a functional group, adding or cleaving a substituent and so on. On the contrary to binding assays some of those assays follow the hydrolysis of a labeled product, e.g. enzyme substrate. According to assay design, a wide variety of different labeling technologies are applied. These assays can utilize a simple labeled substrate, which facilitates the measurement of either substrate or end product, or it may be defined in a way to give direct information of hydrolysis (e.g. internal quenching or energy transfer).
The separation and washing needed in most of these assays make them labor intensive, slow and difficult to automate. Furthermore end point measurement does not allow gathering of kinetic information (e.g. association/dissociation rates). In cases of low affinity bindings, the affinity may be so low that no physical separation can be applied without destroying the binding (e.g. low affinity receptors). Particularly in areas, like screenings (e.g. high throughput screening) there is a constant demand for simpler assays, simplified protocols, which would make automation easier and increase the throughput.
This can be accomplished with homogeneous or nonseparation assays. Homogeneous biomedical assays are defined as assays taking place in one homogeneous phase. It means that no separate phases (such as solid phase catching reagents), and no separation is used prior to measurement. This requires a signal production system that responds to the binding in a way making its direct monitoring possible. Systems known to prior art are e.g. fluorescence polarization assays applied for small molecular compounds, enzyme-monitored immunoassays (Syva Co.), various fluorescence quenching or enhancing assays (for an review see e.g. Hemmila, Applications of Fluorescence in Immunoassays, Wiley, N.Y., 1991). Another category of simplified assay technologies is the nonseparation assays, which, similarly to homogenous assays, avoid separation and washing steps. A good example of such a technology is the scintillation proximity principle marketed by Amersham, which is based on short distance penetration of radiation particles in assay medium and a solid scintillator coated with catching reagents (Udenfriend et al, (1985) Proc Natl Acad Sci, 82, 8672 and Anal Biochem, (1987) 161, 494).
Regardless of a great number of homogeneous assay designs published to day, there are no assays, where the versatility and sensitivity would match those of a good separation assay. The reason to that is manifold relating to e.g. the different way a homogeneous, versus heterogeneous, assay has to be optimized, the control of low affinity nonspecific bindings, and the limitations of applicability of most of the existing homogenous assay techniques. In addition, the conventional homogeneous fluorometric assays are very vulnerable to background interferences derived from various components in the samples. Fluorescence polarizations assays are interfered by low affinity nonspecific bindings (e.g. probe binding to albumin) and autofluorescence of samples.
Energy transfer is a widely used technology to measure and monitor biological reactions. Fluorescence resonance energy transfer (FRET) has been applied e.g. as a spectroscopic ruler in structural studies to measure distances within a macromolecule (Stryer and Haugland (1967) Proc Natl Acad Sci. USA, 58; 719). In addition to resonance energy, transfer, there are other energy transfer reactions, like simple radiative energy transfer (where acceptor absorbs the light emitted by donor), collisional energy transfer, exchange mechanism (Dexter (1953) J Chem Physics, 21, 836), exciton migration (in crystals) and long range electron transfer (Matko et al. 1995, Cytometry 19, 191). In addition, donor emission can be quenched by numerous ways with a number of unrelated compounds having a deactivating effect on some of the donor's energy levels.